NPM1 Mutations Research Articles

The Role of Epithelial-to-Mesenchymal Transition Transcription Factors (EMT-TFs) in Acute Myeloid Leukemia Progression.

Acute myeloid leukemia (AML) is a diverse malignancy originating from myeloid progenitor cells, with significant genetic and clinical variability. Modern classification systems like those from the World Health Organization (WHO) and European LeukemiaNet use immunophenotyping, molecular genetics, and clinical features to categorize AML subtypes. This classification highlights crucial genetic markers such as FLT3, NPM1 mutations, and MLL-AF9 fusion, which are essential for prognosis and directing targeted therapies. The MLL-AF9 fusion protein is often linked with therapy-resistant AML, highlighting the risk of relapse due to standard chemotherapeutic regimes. In this sense, factors like the ZEB, SNAI, and TWIST gene families, known for their roles in epithelial-mesenchymal transition (EMT) and cancer metastasis, also regulate hematopoiesis and may serve as effective therapeutic targets in AML. These genes contribute to cell proliferation, differentiation, and extramedullary hematopoiesis, suggesting new possibilities for treatment. Advancing our understanding of the molecular mechanisms that promote AML, especially how the bone marrow microenvironment affects invasion and drug resistance, is crucial. This comprehensive insight into the molecular and environmental interactions in AML emphasizes the need for ongoing research and more effective treatments.

Mutant NPM1 marginally impacts ribosome footprint in acute myeloid leukemia cells.

NPM1-mutated acute myeloid leukemia (AML) is the most frequent AML subtype. As wild-type NPM1 is known to orchestrate ribosome biogenesis, it has been hypothesized that altered translation may contribute to leukemogenesis and leukemia maintenance in NPM1-mutated AML. However, this hypothesis has never been investigated. We reasoned that if mutant NPM1 (NPM1c) directly impacts translation in leukemic cells, loss of NPM1c would result in acute changes in the ribosome footprint. Here, we performed ribosome footprint profiling (Ribo-seq) and bulk messenger RNA (mRNA) sequencing in two NPM1-mutated cell lines engineered to express endogenous NPM1c fused to the FKBP (F36V) degron tag (degron cells). Incubation of degron cells with the small compound dTAG-13 enables highly specific degradation of NPM1c within 4hours. As expected, RNA-sequencing data showed early loss of homeobox gene expression following NPM1c degradation, confirming the reliability of our model. In contrast, Ribo-seq data showed negligible changes in the ribosome footprint in both cell lines, implying that the presence of NPM1c does not influence ribosome abundance and positioning on mRNA. While it is predictable that NPM1c exerts its leukemogenic activity at multiple levels, ribosome footprint does not seem influenced by the presence of mutant NPM1.

Molecular precision medicine: Multi-omics-based stratification model for acute myeloid leukemia

Acute myeloid leukemia (AML), as the most common malignancy of the hematopoietic system, poses challenges in treatment efficacy, relapse, and drug resistance. In this study, we have utilized 151 RNA sequencing datasets, 194 DNA methylation datasets, and 200 somatic mutation datasets from the AML cohort in the TCGA database to develop a multi-omics stratification model. This model enables comparison of prognosis, clinical features, gene mutations, immune microenvironment and drug sensitivity across subgroups. External validation datasets have been sourced from the GEO database, which includes 562 mRNA datasets and 136 miRNA datasets from 984 adult AML patients. Through multi-omics-based stratification model, we classified 126 AML patients into 4 clusters (CS). CS4 had the best prognosis, with the youngest age, highest M3 subtype proportion, fewest copy number alterations, and common mutations in WT1, FLT3, and KIT genes. It showed sensitivity to HDAC inhibitors and BCL-2 inhibitors. Both the M3 subtype and CS4 were identified as independent protective factors for survival. Conversely, CS3 had the worst prognosis due to older age, high copy number alterations, and frequent mutations in RUNX1, DNMT3A, and TP53 genes. Additionally, it showed higher proportions of cytotoxic cells and Tregs, suggesting potential sensitivity to mTOR inhibitors. CS1 had a better prognosis than CS2, with more copy number alterations, while CS2 had higher monocyte proportions. CS1 showed good sensitivity to cytarabine, while CS2 was sensitive to RXR agonists. Both CS1 and CS2, which predominantly featured mutations in FLT3, NPM1, and DNMT3A genes, benefited from FLT3 inhibitors. Using the Kappa test, our stratification model underwent robust validation in the miRNA and mRNA external validation datasets. With advancements in sequencing technology and machine learning algorithms, AML is poised to transition towards multi-omics precision medicine in the future. We aspire for our study to offer new perspectives on multi-drug combination clinical trials and multi-targeted precision medicine for AML.

Real-Life Management of FLT3-Mutated AML: Single-Centre Experience over 24 Years.

We analyzed 140 patients with a median age of 51 years; 21% had WBC ≥ 100 × 109/L, and 52% had an NPM1 co-mutation. Until 2018, 101 patients received chemotherapy; thereafter, 39 received 3+7+midostaurin. The overall CR rate was 64%, higher in NPM1 mutant patients (73%). Univariate analysis showed that NPM1 mutation (p = 0.032) and WBC < 100 × 109/L (p = 0.013) positively influenced the response, with a trend for FLT3i administration (p = 0.052). Multivariate analysis confirmed WBC count as an independent prognostic factor (p = 0.017). In CR1, 41/90 patients underwent allogeneic and 18 autologous transplantation. The median EFS was 1.1 vs. 1.6 years in autografted and allografted patients, respectively (p = 0.9). The one-year non-relapse mortality was 0.00% for autologous and 28% for allogeneic transplants (p = 0.007); CIR at 1 and 3 years was higher in autologous transplants (39% vs. 15% and 57% vs. 21%, p = 0.004). The median survival was not reached in the FLT3i group. Overall, 69 patients received stem cell transplantation (18 autologous, 51 allogeneic). Post-transplant FLT3i was resumed in eight patients, all alive after a median of 65 months. Allogeneic transplantation is crucial in FLT3-mutated AML, but the next challenge will be to identify which patients can benefit from transplants in CR1 and in which to intensify post-transplant therapy.

Human Leukocyte Antigen-DR and Cluster Designation 34: Double-negative Acute Myeloid Leukemias – Our Experience in Clinical Setting

Abstract Introduction: Dual negativity for cluster designation (CD) 34 and human leukocyte antigen (HLA)-DR antigen in leukemia panel is an important finding in clinical practice. The combination of this finding though seen classically in acute promyelocytic leukemia (APL) is not specific to it but can be seen in many other myeloid leukemias like the ones associated with nucleophosmin 1 (NPM1) or FMS-related tyrosine kinase 3 gene-internal tandem duplication (FLT3-ITD) abnormalities. It is important to identify APL from non-APL leukemia as treatment and prognosis vary. Aims and Objectives: We analyzed CD 34 and HLA-DR dual-negative acute myeloid leukemia (AML) cases presented to us over 2.5 years (26 cases) and segregated them into APL and non-APL groups to study their morphological/flow cytometric and cytogenetic profile and correlation with treatment response. Materials and Methods: It was a prospective study including all newly diagnosed AMLs showing CD 34 and HLA-DR dual negativity in flow cytometry profile. Cases in which complete information/cytogenetics/molecular profile were not available, were excluded from the study. The patients were followed up till the end of induction to look for morphological response to induction therapy. The clinical and treatment records were pulled out from the patient database after ethical committee clearance. Results: A total of 139 new AML cases were encountered during the study period out of which 28 (20%) were found to be dual negative for CD34 and HLA-DR. The non-APL group showed higher mean age, TLC at baseline, variable morphology, and more number of aberrant expression on flow cytometry profile. No significant difference was noted in terms of gender, presence of hepatosplenomegaly, DIC as complication, or percentage of blasts/blasts equivalent at baseline. Our study found that while all APL patients had PML-RARA, the associated molecular abnormalities were much lesser as compared to the non-APL group which showed a variety of abnormalities such as NPM1, FLT3, RAS mutation, and mutations involving epigenetic modifiers. The response rate to induction therapy was significantly lower in the non-APL group as compared to the APL group. Conclusion: The non-APL myeloid leukemia which is dual negative is commonly found to be associated with cup-shaped morphology, NPM, or FLT3-ITD mutation along with other abnormalities. These patients were older in age, showed higher TLC and higher blast counts, associated with poorer response to induction therapy as compared to the APL group.

Exploration of ETV6::ABL1-positive AML with concurrent NPM1 and FLT3-ITD mutations.

ETV6::ABL1 is a rare fusion gene that found in MPN, ALL, and AML. It has a complex and diverse formation mechanism due to the reciprocal orientations of the ETV6 and ABL1 genes relative to the centromeres. NPM1 is frequently mutated in adult AML, often accompanied by FLT3-ITD, which suggests molecular synergisms in AML pathogenesis. Previous reports on ETV6::ABL1 mostly focus on FLT3-ITD. In this study, we present a case of AML with ETV6::ABL1, along with NPM1 and FLT3-ITD. The patient showed a rapid increase in primitive cells at the initial stage, along with the presence of immature granulocytes and erythrocytes. Through cytogenetic analysis, fluorescence in situ hybridization (FISH), and RNA-seq, we elucidated the mechanism behind the formation of the ETV6::ABL1 fusion gene. Despite conventional chemotherapy failure and rapid tumor proliferation, we attempted to add FLT3 inhibitor sorafenib to the treatment, along with chemotherapy bridging to haploidentical transplantation. After haplo-HSCT, a combination of sorafenib and dasatinib was administered as maintenance therapy. The patient achieved complete remission (CR) and maintained it for 11 months. The intricate genetic landscape observed in this case presents diagnostic dilemmas and therapeutic challenges, emphasizing the importance of a comprehensive understanding of its implications for disease classification, risk stratification, and treatment selection.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a proapoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2 dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c+ cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment of the AKT/mTORC1 pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

Characteristic Analysis of Adult Acute Myeloid Leukemia Patients with PTPN11 Gene Mutation

To investigate the incidence of PTPN11 gene mutation and its associated gene mutations in adult patients with acute myeloid leukemia (AML), and analyze its clinical characteristics. Second-generation sequencing and Sanger sequencing were used to detect 51 gene mutations, and multiplex-PCR was used to detect 41 fusion genes from 451 newly diagnosed adult AML patients admitted to Affiliated Hospital of Jiangnan University, Changzhou Second People's Hospital, Wuxi People's Hospital and Wuxi Second People's Hospital from January 2017 to July 2022. Among 451 primary adult AML patients, the PTPN11 gene mutation was detected in 34 cases, and the mutation rate was 7.5%. In the 34 patients, 37 PTPN11 alterations were found, which were exclusively missense mutations affecting residues located within the N-SH2 (31 cases) and PTP (6 cases) domains and clustered overwhelmingly in exon 3. The platelet count of PTPN11 mutation patients was 76.5(23.5, 119.0)×109/L, which was significantly higher than 41.0(22.0, 82.5)×109/L of wild-type patients (P < 0.05). While, there were no significant differences in sex, age, peripheral white blood cell count, hemoglobin, and bone marrow blast between PTPN11 mutation and wild-type patients (P >0.05). In FAB subtypes, PTPN11 mutations were mainly distributed in M5, followed by M2 and M4, less frequently in M3 and M6. There was no significant difference in the distribution of FAB subtypes between PTPN11 mutation and wild-type patients (P >0.05). A total of 118 AML patients were detected positive fusion gene, among which patients with PTPN11 mutations had a higher incidence of positive MLL-AF6 than wild-type ones (P < 0.01). 97.1% of 34 patients with PTPN11 mutations were accompanied by other mutations, in descending order, they were respectively NPM1 (38.2%), NRAS (32.4%), FLT3-ITD (32.4%), DNMT3A (32.4%) and KRAS (23.5%), etc . PTPN11 mutation has a certain incidence in AML patients and is clustered overwhelmingly in exon 3. ALL of them are exclusively missense mutations, and most often present in conjunction with NPM1 mutations. FAB typing of PTPN11 mutation is mostly manifested as M5 subtype, which is associated with higher platelet counts.

Outcomes of Patients With Newly Diagnosed AML and Hyperleukocytosis.

AML presenting with hyperleukocytosis is associated with poor outcomes. We aim to understand the factors associated with early mortality and overall survival (OS) to help guide management and improve early mortality. We retrospectively reviewed data from 129 consecutive patients with newly diagnosed AML and a WBC count ≥100 × 109/L between January 2010 and April 2020. Logistic regression models estimated odds ratios for 4-week mortality. Cox proportional hazard models estimated hazard ratios for OS. The median age was 65 years (range, 23-86); the median WBC was 146 × 109/L (range, 100-687). Seventy-five (58%) patients had clinical leukostasis (CL). FLT3, NPM1, and RAS pathway mutations were detected in 63%, 45%, and 27% of patients, respectively. Cytoreduction consisted of hydroxyurea in 124 (96%) patients, cytarabine in 69 (54%), and leukapheresis in 31 (24%). The cumulative 4-week and 8-week mortality rates were 9% and 13%, respectively, all in patients age 65 years and older. By multivariate analysis, older age, CL, and thrombocytopenia <40 × 109/L were independently associated with a higher 4-week mortality rate. After a median follow-up of 49.4 months, the median OS was 14.3 months (95% CI, 7 to 21.6), with 4-year OS of 29%. Age 65 years and older, CL, tumor lysis syndrome, elevated LDH ≥2,000 U/L, elevated lactate ≥2.2 mmol/L, and poor-risk cytogenetics were independent factors associated with worse OS. Hyperleukocytosis is a life-threatening hematologic emergency. Early recognition and intervention including cytoreduction, blood product support, antibiotics, and renal replacement therapy may help mitigate the risk of morbidity and early mortality.

Description of lymphocyte and cytokine profiles in individuals with acute myeloid leukemia associated with FLT3-ITD and NPM1 mutation status.

The pathogenesis of acute myeloid leukemia (AML) involves mutations in genes such as FLT3 and NPM1, which are also associated with the prognosis of the disease. The immune system influences disease progression, but the mechanisms underlying the interaction between the immune system and AML are not clear. In this study, the profiles of lymphocytes and cytokines were described in individuals with AML stratified by molecular changes associated with prognosis. The participants included in this study were newly diagnosed AML patients (n = 43) who were about to undergo chemotherapy. Subtypes of lymphocytes in peripheral blood, including B cells, T cells, and natural killer cells, and serum concentrations of cytokines, including Th1, Th2, and Th17, were studied by flow cytometry assays (BD FACSCanto II). The correlations between lymphocyte subsets, cytokines, and genetic/prognostic risk stratification (based on the FLT3 and NPM1 genes) were analyzed. The differences in B lymphocytes (%), T lymphocytes (%), plasmablasts (%), leukocytes (cells/µl), and tumor necrosis factor (pg/ml) were determined between groups with FLT3-ITD+ and FLT3-ITD- mutations. The presence of mutations in NPM1 and FLT3-ITD and age suggested changes in the lymphocyte and cytokine profile in individuals with AML.

Do NPM1 and FLT3-ITD mutations modify prognosis in patients treated with non-intensive regimens?

FLT3-ITD and NPM1 mutations are key to defining the genetic risk profile of acute myeloid leukemia (AML). We aimed to assess the prognostic features of the FLT3-ITD and NPM1 mutations in old and/or unfit individuals with AML treated with non-intensive therapies in the era before azacitidine-venetoclax approbation. The results of various non-intensive regimens were also compared. We conducted a retrospective analysis that included patients treated with different non-intensive regimens, between 2007 and 2020 from PETHEMA AML registry. We compiled 707 patients with a median age of 74years and median follow-up time of 37.7months. FLT3-ITD patients (N = 98) showed a non-significant difference in overall survival (OS) compared to FLT3-ITD negative-patients (N = 608) (P = 0.17, median OS was 5 vs 7.3months respectively). NPM1-mutated patients (N = 144) also showed a non-significant difference with NPM1 wild type (N = 519) patients (P = 0.25, median OS 7.2 vs 6.8 respectively). In the Cox regression analysis neither NPM1 nor FLT3-ITD nor age were significant prognostic variables for OS prediction. Abnormal karyotype and a high leukocyte count showed a statistically significant deleterious effect. Azacitidine also showed better survival compared to FLUGA (low dose cytarabine plus fludarabine). NPM1 and FLT3-ITD seem to lack prognostic value in older/unfit AML patients treated with non-intensive regimens other than azacitidine-venetoclax combination.

Multi-target measurable residual disease assessed by error-corrected sequencing in patients with acute myeloid leukemia: An ALFA study

The evaluation of measurable residual disease (MRD) in acute myeloid leukemia (AML) using comprehensive mutation analysis by next-generation sequencing (NGS) has been investigated in several studies. However controversial results exist regarding the detection of persisting mutations in DNMT3A, TET2, and ASXL1 (DTA). Benchmarking of NGS-MRD taking into account other molecular MRD strategies has to be done. Here, we performed error-corrected-NGS-MRD in 189 patients homogeneously treated in the ALFA-0702 study (NCT00932412). Persistence of non-DTA mutations (HR = 2.23 for RFS and 2.26 for OS), and DTA mutations (HR = 2.16 for OS) were associated with poorer prognosis in multivariate analysis. Persistence of at least two mutations in complete remission (CR) was associated with a higher cumulative incidence of relapse (CIR) (HR = 3.71, p < 0.0001), lower RFS (HR = 3.36, p < 0.0001) and OS (HR = 3.81, p = 0.00023) whereas persistence of only one mutation was not. In 100 analyzable patients, WT1-MRD, but not NGS-MRD, was an independent factor for RFS and OS. In the subset of 67 NPM1 mutated patients, both NPM1 mutation detection (p = 0.0059) and NGS-MRD (p = 0.035) status were associated with CIR. We conclude that detectable NGS-MRD including DTA mutations correlates with unfavorable prognosis in AML. Its integration with alternative MRD strategies in AML management warrants further investigations.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT’s adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Abstract IA023: Targeting epigenetic complexes in leukemia

Abstract Chromatin-associated multi-protein complexes, which often comprise proteins involved in epigenetic regulation, play an important role in various cancers, including leukemia. Thus, pharmacologic inhibition of proteins involved in these complexes can lead to new anti-cancer therapies. We have focused on therapeutic targeting of various chromatin-bound multi-protein complexes, including Mixed Lineage Leukemia 1 (MLL1) and Polycomb Repressive Complex 1 (PRC1) complexes, as a novel treatment for leukemia. These efforts led to the development of small molecule inhibitors targeting the menin-MLL1 interaction, which were translated to leukemia patients, demonstrating promising efficacy in Acute Myeloid Leukemia (AML) with NPM1 mutations or MLL1 translocations. However, clinical studies revealed resistance to the single agent menin inhibitor in a sub-set of AML patients, suggesting that combinatorial treatments or new generations of menin inhibitors might be required to overcome resistance. Our recent efforts to address resistance to menin inhibitors will be presented, including combinatorial studies with other targeted agents (e.g. kinases inhibitors), which resulted in synergistic effects. Furthermore, discovery of a new generation of menin inhibitors with promising activity against menin patient mutations will also be presented. Citation Format: Jolanta Grembecka. Targeting epigenetic complexes in leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA023.

Co-mutation landscape and its prognostic impact on newly diagnosed adult patients with NPM1 mutated de novo acute myeloid leukemia.

e18505 Background: 25-35% of adult patients with acute myeloid leukemia (AML) carries NPM1 mutation, characterized by aberrant cytoplasmic dislocation of NPM1 protein, which is generally associated with a favorable outcome in the absence of FLT3-ITD mutation. Indeed, for NPM1-mutated AML patients with different co-mutation profiles, identification of specific co-mutation combinations other than FLT3-ITD mutation is necessary for accurate risk stratification and contributes to identify the optimal population who may benefit from allogeneic hematopoietic stem cell transplantation (allo-HSCT) during post-remission therapy. Methods: Eligible patients included newly diagnosed adult patients (≥18 years old) with NPM1 mutated de novo AML (M3 excluded) treated at the First Affiliated Hospital of Zhejiang University School of Medicine for at least one complete course of induction therapy from October 2018 to December 2022. All patients received next generation sequencing (NGS) detection for diagnosis before treatment. Response was evaluated within two courses of induction chemotherapy. Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were collected for endpoint analysis. Results: 178 patients were analyzed. Median age was 59 years old (IQR, 51-68). The co-mutated genes with a detection rate of ≥ 10% included FLT3, DNMT3A, TET2, IDH2, IDH1, PTPN11, NRAS, and WT1. The median follow-up was 26.2 months. Our results showed that in NPM1 mutated AML, co-mutations of IDH1, IDH2, and PTPN11-PTP domain were associated with favorable prognosis, while co-mutations of FLT3-ITD and DNMT3A were associated with poor prognosis. Interestingly, the adverse outcome trends brought by FLT3-ITD and DNMT3A co-mutations could be reversed by IDH mutation and PTPN11 mutations, respectively (Table). Besides, for NPM1 mutated AML patients combined with FLT3-ITD/ DNMT3A mutations, allo-HSCT after remission could significantly improve outcome ( P &lt; 0.05), while similar results were not found in FLT3-ITD/ DNMT3A wild type patients. Conclusions: This study demonstrated that NPM1-mutated AML with different co-mutation profiles led to heterogeneous outcomes. For NPM1-mutated AML patients with adverse prognosis-related co-mutation combinations, allo-HSCT after remission may be a recommended strategy to improve outcome. [Table: see text]

Venetoclax combined with hypomethylating agents for favorable risk acute myeloid leukemia: A single center experience.

e18514 Background: Favorable risk acute myeloid leukemia (FR-AML) is typically treated with intensive induction chemotherapy followed by high dose consolidative cytarabine. However, a subgroup of FR-AML are not candidates for high intensity induction because of advanced age, comorbidities, or poor performance status. For these patients, hypomethylating agents (HMA) combined with venetoclax (HMA-Ven) is inferred as the standard of care based upon results of the VIALE-A trial; however, that trial enrolled only intermediate or poor risk AML. The efficacy of HMA-Ven in FR-AML is unclear. Here we present a single center experience of this regimen in FR-AML. Methods: In this retrospective single center case series, we collected data from all patients &gt;18 years old with newly diagnosed FR-AML between January 2018 to December 2022 at AdventHealth Orlando who were treated with HMA-Ven. Patient with relapsed/refractory disease or a diagnosis of acute promyelocytic leukemia were excluded. FR-AML was defined using ELN 2022 guidelines based on the results of genomic testing of the initial bone marrow aspirate. The primary endpoints were the complete remission (CR) rate and 1-year overall survival (OS). Statistical analyses were performed using the R survminer package. Results: Newly diagnosed FR-AML who received HMA-Ven were identified (n=12). The median age was 67.5 years. 3 (25%) cases had inversion of chromosome 16 and 9 (75%) had NPM1 mutations without FLT3-ITD mutations. 11 (92%) patients received decitabine and venetoclax. Patients received a median of 2.5 cycles of HMA-Ven, and all 12 patients achieved CR after one cycle of treatment. 6 (50%) patients were consolidated with intermediate-dose cytarabine (IDAC), 3 (25%) continued HMA-Ven, 2 (17%) underwent allogeneic stem cell transplant, and 1 (8%) received enasidenib. The 1-year OS was 80% (CI 0.59-1), and the median OS was not reached. 1 patient died due to chemotherapy toxicity with IDAC, and 1 patient died due to heart complications. 2 patients relapsed, 1 after IDAC and 1 after intolerance to further treatment after 6 cycles of HMA-Ven. Conclusions: Our findings suggest that HMA-Ven is an effective treatment for newly diagnosed favorable risk AML. All patients achieved CR with one cycle of treatment with most surviving more than one year. Large multicenter studies are needed to further validate these promising results.

RNA expression-based risk stratification of de novo acute myeloid leukemia.

e18503 Background: Prognostication of acute myeloid leukemia (AML) at initial diagnosis relies on the identification of certain underlying genetic abnormalities as proposed by the European LeukemiaNet (ELN) risk classification. However, AML is highly heterogenous at the molecular level and standard guidelines are often not straightforwardly applicable. In this study, we explored gene expression-based risk stratification strategies and found that FLT3 and NPM1 transcript level profiles can predict overall survival in de novo AML. Methods: The Cancer Genome Atlas AML (TCGA-LAML) project’s normalized RNA-Seq transcriptome profile (n=158) published in 2013 was analyzed with MATLAB software. "High” or “low” level of each gene was determined by the median RPKM of the cohort used as cutoff. P-values were obtained from Cox proportional hazards regression model. Results: The low-FLT3 level group was associated with superior median OS (mOS) compared to high-FLT3 group (24 vs. 16 months; p = 0.02), whereas high-NPM1 level group was associated with superior mOS compared to low-NPM1 group (26 vs. 12 months; p = 0.03). The mOS for high-FLT3/low-NPM1 and low-FLT3/high-NPM1 groups were 9 months and 27 months, respectively (p = 0.001). In high-FLT3/low-NPM1 and low-FLT3/high-NPM1 groups, composition of Cancer and Leukemia Group B (CALGB) cytogenetic risk category [favorable, intermediate, adverse] was [9%, 73%, 18%] and [32%, 49%, 19%], respectively; composition of French-American-British (FAB) classification [M0, M1, M2, M3, M4, M5, M6, M7] was [15%, 27%, 3%, 6%, 24%, 24%, 0%, 0%] and [3%, 14%, 41%, 19%, 22%, 0%, 0%, 3%], respectively; composition of FLT3/NPM1c mutations [ wt/wt, mut/wt, wt/mut, mut/mut, NOS] was [45%, 18%, 18%, 18%, 0%] and [61%, 14%, 7%, 14%, 2%], respectively. Conclusions: We demonstrate a de novo AML risk categorization method solely based on FLT3 and NPM1 RNA-Seq transcript levels. The mOS of high-FLT3/low-NPM1 group resembled that of ELN "adverse" risk group, whereas the mOS of low-FLT3/high-NPM1 group resembled that of ELN "favorable" risk group. High-FLT3/low-NPM1 group was composed of fewer "favorable” but more “intermediate” CALGB risk categories compared to low-FLT3/high-NPM1 group. Notably, considerably more FAB M0, M1 and M5 but fewer M2 and M3 classes were enriched in high-FLT3/low-NPM1 group compared to low-FLT3/high-NPM1 group. We also note approximately half of the cases analyzed were FLT3 and NPM1 wild-types and speculate FLT3 and NPM1 transcript profiles may represent a global oncogenic state regardless of the underlying FLT3 or NPM1 mutations. This is also supported by the previously reported FLT3 mutation-like transcriptomic profiles observed in FLT3 wild-type AML cases1). Reference: 1) Mosquera Orgueira A, et al. Gene expression profiling identifies FLT3 mutation-like cases in wild-type FLT3 acute myeloid leukemia. PLoS One. 2021 Feb 16;16(2):e0247093

Genomic evolution of patients with myeloid neoplasms and known antecedent clonal hematopoiesis.

6584 Background: Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem cells (HSCs) with one or more somatic mutations in the absence of a hematologic malignancy. Although it is associated with normal aging, CH confers an increased risk of several comorbidities and hematologic malignancies, including myeloid neoplasms (MNs). However, the outcomes of patients with CH that transformed to MN are unknown. Here, we aim to evaluate the clinicopathologic characteristics and survival outcomes of patients with MN with known antecedent CH. Methods: We retrospectively evaluated patients seen at a tertiary cancer center from May 2016 to July 2023 with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) and identified those who had CH prior to MN diagnosis. At the time of MN transformation, genomic data was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Results: A total of 36 patients were identified; at the time of CH diagnosis, 79% had known cardiovascular comorbidities and 81% had a prior diagnosis of non-myeloid malignancies for which 72% received prior chemotherapy and 25% radiation. According to the Clonal Hematopoiesis Risk Score (CHRS), 22% were low, 56% deemed intermediate, and 22% at high risk of transformation to MNs. The most frequently observed CH-associated mutations were TET2 (41%), TP53 (38%), IDH2 (16%), EZH2 (16%), ASXL1 (13%), and DNMT3A (13%); 69% had more than one somatic mutation, and 61% had normal cytogenetics. The median time to transformation to MN was 15 months, 26 months, and 24 months for MDS (n=28), CMML (n=2), and AML (n=6) patients, respectively (p=0.72). The median overall survival from MN diagnosis was 54 months, not reached, and 2 months for MDS, CMML, and AML, respectively (p=0.0015). We further delved into the genomic evolution from CH to MN. In the 6 pts who transformed to AML, 4 had a TP53 mutation at CH with expansion of the clone at MN transformation, 2 acquired NPM1 mutations and 1 acquired FLT3 mutations. Both pts who progressed to CMML had expansion of their underlying CH clones (KRAS and ZRSR2). In those who developed MDS, 22 pts had expansion of their CH-mutated clone at the time of MN, most frequently in TET2 (36%), IDH2 (18%), and TP53 (14%); 5 pts acquired new driver mutations outside of their CH clone; and 1 pt lost their CH clone with no new somatic mutations detected. Conclusions: Individuals with CH are at risk of progressing to MN, especially with external selective pressures like antineoplastic therapy that are not accounted for in prognostic systems like CHRS. Patients often have underlying high-risk clones at the time of CH diagnosis that expand at the time of MN, though some patients acquired new driver mutations.

Latest results of a phase 2 study of IMM01 combined with azacitidine (AZA) as the first-line treatment in adults with higher risk myelodysplastic syndromes (MDS).

6510 Background: Patients diagnosed with higher-risk myelodysplastic syndrome (MDS) have poor prognosis. Azacitidine (Aza) has been shown to prolong survival in patients with treatment-naive, higher-risk MDS compared to a menu of standard of care options. IMM01 is a fusion protein comprising a recombinant signal regulatory protein α (SIRPα) and IgG1, exerting anti-tumor effects by inhibiting the "Don't eat me" signal and activating the "Eat me" signal, leading to robust antibody-dependent cellular phagocytosis (ADCP). Methods: This is an open-label, multi-center, phase 2 study (NCT05140811) that evaluated safety and efficacy of IMM01 in combination with AZA as the first-line treatment for patients with untreated higher-risk MDS. Enrolled patients were aged ≥18 years with intermediate to very high risk MDS, as defined by the Revised International Prognostic Scoring System (IPSS-R) score &gt;3.5, and were not eligible for stem cell transplant or intensive chemotherapy. Patients were received intravenous IMM01 at a dosage of 2.0mg/kg/week and subcutaneous AZA at a dosage of 75 mg/m2 on days1-7 per 28-day cycle. Results: By the cutoff-day of Dec 22, 2023, 57 patients were enrolled in the study. The median age was 64 (30-83) years, with 41 (71.9%) being male, and 55 (96.5%) having an ECOG of ≥1. Based on risk classification per IPSS-R, 43.9% were high risk (HR) and 31.6% were very high risk (vHR). At baseline, the median levels of blood counts were 69 (35-136)g/L for hemoglobin, 43 (2-409)×109/L for platelets and 0.8 (0.1-8.6)×109/L for neutrophils. The median duration of follow-up was 12.8 months (95%CI:9.7-15.3). Among the 51 efficacy evaluable patients, overall response rate (ORR) was 64.7%, including 29.4% complete response (CR) rate, 15.7% marrow CR (mCR) with hematologic improvement (HI), 5.9% HI and 13.7% mCR alone. The median time to response (TTR) was 1.9 months (95%CI:1.8-2.8) and the median duration of response (DoR) was not reached(NR). The median of progression-free survival (PFS) was not reached, with an estimated 12-month PFS of 54.4% (95% CI, 31.4-72.6). NGS analysis identified common mutations in DNMT3A, ASXL1, U2AF1 and RUNX1. Notably, NPM1 mutations significantly correlate with treatment response, particularly achieving CR. The most common ≥G3 treatment related adverse events (TRAEs) (≥10%) included leukopenia (78.9%), thrombocytopenia (66.7%), neutropenia (66.7%), lymphopenia (56.1%), anemia (43.9%), infection (15.8%) and pneumonia (10.5%). Without using of a low priming dose, the Grade ≥3 hemolysis was rare (only 1.8%). Conclusions: IMM01 (without low-dose priming) combined with AZA were well tolerated and showed exciting efficacy results in patients with treatment-naive higher-risk MDS. Clinical trial information: NCT05140811 .

Chromothripsis in myeloid malignancies.

Chromothripsis refers to massive genomic rearrangements developed during a catastrophic event. In total acute myeloid leukemia (AML), the incidence of chromothripsis ranges from 0 to 6.6%, in cases of complex karyotype AML, the incidence of chromothripsis ranges from 27.3 to 100%, whereas in cases of AML with TP53 mutations, the incidence ranges from 11.1 to 90%. For other types of malignancies, the incidence of chromothripsis also varies, from 0 to 10.5% in myelodysplastic syndrome to up to 61.5% in cases of myelodysplastic syndrome with TP53 mutations.Chromothripsis is typically associated with complex karyotypes and TP53 mutations, and monosomal karyotypes are associated with the condition. ERG amplifications are frequently noted in cases of chromothripsis, whereas MYC amplifications are not. Moreover, FLT3 and NPM1 mutations are negatively associated with chromothripsis. Chromothripsis typically occurs in older patients with AML with low leukocyte counts and bone marrow blast counts. Rare cases of patients with chromothripsis who received intensive induction chemotherapy revealed low response rates and poor overall prognosis. Signal pathways in chromothripsis typically involve copy number gain and upregulation of oncogene gene sets that promote cancer growth and a concomitant copy number loss and downregulation of gene sets associated with tumor suppression functions.Patients with chromothripsis showed a trend of lower complete remission rate and worse overall survival in myeloid malignancy. Large-scale studies are required to further elucidate the causes and treatments of the condition.